PROJECT SUMMARY/ABSTRACT The intestinal epithelium is comprised of a single epithelial layer with both absorptive and secretory functions and is responsible for nutrient absorption. Additionally it is in constant contact with the abundant and diverse indigenous microbiota that inhabits the intestinal lumen. The epithelium and the microbiota exists in a remarkably stable, bal- anced symbiosis. This symbiosis is responsible for homeostasis of the intestinal ecosystem, and disturbances in this relationship has been associated with a wide variety of disease states. While animal models and the use of germ-free and gnotobiotic mice have led to remarkable advances in our understanding of host-microbial interac- tions, species-specific differences have made it difficult to study many human enteric pathogens, and suitable in vitro cell culture models that accurately represent human physiology are severely lacking. We have recently de- scribed an in vitro system that accurately reflects both the complex cellular makeup and the topological organization of the human intestine. Using human pluripotent stem cells (hPSCs), we have generated 3-dimensional (3D) intes- tinal units called Human Intestinal Organoids (HIOs). HIOs contain both intestinal epithelium and mesenchyme and are comprised of all of the different cell types found in the small intestine. HIOs have already proven to be a powerful system to study human intestinal development. Our preliminary results demonstrate that HIOs are also an excellent system to study how enteric pathogens affect the host epithelium. Therefore, the overarching goal of this project is to determine how specific enteric pathogens and normal microbiota affect the host HIO epithelium at the cellular and molecular level, and to determine how complex host-microbiota-pathogen relationships are estab- lished and maintained within the HIO. We propose to 1) investigate how interaction between commensal microbes and the HIO epithelium affects each partner in this symbiosis, 2) determine how contact pathogens affects the physiology of the HIO epithelium and 3) determine if contact with commensal microbes will alter the patho- gen/epithelium interaction. To accomplish these specific aims, we have established a multidisciplinary team that will examine the complex relationship between the HIO epithelium, the indigenous microbiota and viral and bacterial enteric pathogens.